Microbiology Research 2013; volume 4:e4

Abstract

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Komodo dragons (Varanus komodoensis)

are able to feed on large prey items by injecting a dose of toxic bacteria with their bite that,
over time, kills the prey by systemic infection.
Dragons also suffer bites from other members
of their own species during territorial disputes
and feeding frenzies. However, they do not suffer the same fate as their prey, suggesting that
they have developed a strong immunity to bacterial infections. This study was undertaken to
determine the antibacterial activities of serum
from the Komodo dragon. Bacterial cultures
were treated with different volumes serum
from Varanus komodoensis and the growth was
monitored by optical density at 430 nm. In
addition, the serum was treated with protease,
chelators of divalent metal ions, or with mild
heat to determine the mechanism of antibacterial activities. Treatment of bacterial cultures with serum from Komodo dragons
(Varanus komodoensis) resulted in a volumedependent decrease in bacterial growth.
Cultures of Escherichia coli, Staphylococcus
aureus, and Klebsiella oxytoca exhibited moderate-strong growth inhibition by V. komodoensis serum, while cultures of Streptococcus epidermitis, Salmonella typhimurium, Providencia stuartii, and Shigella flexneri were nearly
completely obliterated for 24 h by only 10%
(v/v) serum. The antibacterial activity of V.
komodensis serum occurred very rapidly, as
18% of E. coli growth was inhibited by a five
min exposure to serum. Furthermore, 10- and
20-min incubations of E. coli with serum from
V. komodoensis resulted in 43 and 68% inhibition of bacterial growth, respectively. The bactericidal capacity of the serum against E. coli
was 2,075,000 bacteria/L serum, and was
inhibited by mild heat treatment, pronase,
EDTA, and phosphate, indicating that the antibacterial action is most probably due to the
presence of a potent serum complement protein system.

[page 16]

Acknowledgements: the authors wish to acknowledge the help of San Antonio Zoo employees: J.
Stephen McCusker (Director), Alan Kardon
(Curator of Herpetology and Aquarium), and Rob
Coke and Jenny Nollman (Veterinarians), and
Houston Zoo employees Rick Barongi (Director),
Stan Mays (Director of Herpetology), and
Maryanne Tocidlowski (Veterinarian), for permission to work with zoo animals and collection
of samples. In addition, we also thank technicians Courtney Threadgill, Candice Robinson,
and Herpetology/Aquarium staff (San Antonio
Zoo) for help in restraining animals during blood
collection.

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of Chemistry, McNeese
State University, Lake Charles, LA;
2Department of Herpetology, San
Antonio Zoo, TX; 3Department of
Herpetology, Houston Zoo, TX, USA

Key words: innate immunity, lizard, reptile,

serum complement, varanid.

Contributions: MM, provided grant funding,

designed the experiments for the study, conducted some of the laboratory work, wrote the majority of the manuscript; DHRF conducted the majority of the laboratory work, wrote portions of the
manuscript; BM, JB, conceived of the general
idea of the study, coordinated the collection of
samples, made small contributions to the development of the manuscript.

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1Department

is an endangered species of monitor lizard

indigenous to only five islands in the Lesser
Sunda region of southeast Indonesia.1 It is the
largest lizard in the world, reaching lengths of
more than 3 meters.2 These reptiles primarily
inhabit the tropical savannahs, and their
boundary woodlands, of these islands. They
feed as both scavenger and predators,3 eating a
broad spectrum of food items.
Komodo dragons kill prey items with a lethal
bite that is believed to inject toxic bacteria into
the blood stream of these animals.4-5 However,
recent studies have shown that Komodo dragon saliva also contains a potent and complex
venom, which causes anticoagulation and
induction of systemic shock in prey animals.6
After days, or even weeks, depending on the
size of the prey and the number of bites, the
prey succumbs to the lethal effects of systemic
infection or shock. However, despite the fact
that Komodo dragons fight and inflict bites on
each other,2 these lizards are not known to be
affected by septic infections. This would seem
to indicate that these animals have developed
some type of immune mechanism(s) that protect against potential sepsis due to bites from
other Komodo dragons.
Despite the endangered status of the
Komodo dragon, practically nothing is known
about the immune systems of these threatened
reptilians. Knowledge of the immune system
will be of interest to both evolutionary biologists and conservationists of these reptilians.
The primary goal of this project was to characterize the antibacterial properties of serum
from the Komodo dragon.

Mark Merchant,1 Danyell Henry,1

Rodolfo Falconi,1 Becky Muscher,2
Judith Bryja3

Correspondence: Mark Merchant, DEof Biochemistry, McNeese State University

Lake Charles, LA 70609, USA. Tel. +1.337.4755773
E-mail: mmerchant@mcneese.edu

Antibacterial activities
Introduction
of serum from the Komodo
Dragon (Varanus komodoensis) The Komodo dragon (Varanus komodoensis)

Materials and Methods

Chemicals and biochemicals

Nutrient broth and low EEO agarose were

purchased from Intermountain Scientific
(Kaysville, UT). Bacterial cultures (American
Type Culture Collection) were purchased from
Remel (Lenexa, KS). The following species
were used for representatives of Gram- negative species: Escherichia coliform (ATCC
25922), Shigella flexnerii (ATCC 700930), and
Klebsiella oxytoca (ATCC 49131), Salmonella
typhimurium (ATCC 14028), Providencia stuartii (33672) and the following Gram- positive
species: Staphylococcus aureus (ATCC 6538),
and Streptococcus epidermitis (ATCC 19615).
Pronase from Streptomyces griseus was purchased from Sigma Chemical Company (St.
Louis, MO).

[Microbiology Research 2013; 4:e4]

Funding: this research was funded by a McNeese

State University Alumni Association Faculty
Development Award. In addition, support was provided by a NSF Research Commercialization/
Educational Enhancement Plan grant administered by the Louisiana Board of Regents.
Conflict of interests: the authors declare no
potential conflict of interests.
Received for publication: 30 August 2011.
Revision received: 14 August 2012.
Accepted for publication: 29 August 2012.
This work is licensed under a Creative Commons
Attribution NonCommercial 3.0 License (CC BYNC 3.0).
Copyright M. Merchant et al., 2013
Licensee PAGEPress, Italy
Microbiology Research 2013; 4:e4
doi:10.4081/mr.2013.e4

Collection of blood samples

Blood samples were collected from Komodo
dragons at the Houston and San Antonio zoos.
Blood was drawn from the tail vein, transferred
to Vaccutainer tubes, and allowed to clot for
at least five h at ambient temperature before
the serum was collected. The amount of blood
collected from each individual depended on the
size of the animal, and was at the discretion of

Article

Kinetic antibacterial assay

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Five hundred L of Komodo dragon serum

were inoculated with 20 L of an E. coli culture
in log phase growth. At various time points, 50
L of a dilution of each sample were spread
onto the surface of nutrient broth agar plates
to determine the CFUs for each sample. The
samples were typically plated at five different
dilutions (1:100-1:106, in sterile isotonic
saline) to obtain plates with a quantity of
colonies such to provide a reasonable estimate
of bacterial density (50-400 CFUs per plate).
To determine possible mechanisms of antibacterial action, pooled serum from Komodo dragons was incubated with one unit/mL pronase
(isolated from Streptomyces griseus), 5 mM
EDTA (pH 8.0), or 5 mM phosphate for 30 min
at ambient temperature. In addition, four
aliquots of serum were incubated at 56oC for
30 min. These serum samples (500 L) were
all incubated with 20 L of an E. coli culture in
log phase growth. The samples were incubated
for 15 min at ambient temperature, diluted
serially (1:100 -1:106) in sterile saline, and 50
L of each sample were plated on nutrient
agarose as described above.

Statistics and controls

All assays were conducted in quadruplicate,
and the results are expressed as the means
standard deviations for four independent replicates. The statistical comparisons between
groups were conducted using analyses of variance and Duncans post-hoc comparisons and
P<0.05 was chosen as the standard probability
of statistical significance.

The data in Table 1 exhibits the results of a

mechanistic study, showing the susceptibility
of the antibacterial effects to mild heat treatment and chelators of divalent metal ions.
Treatment of E. coli cultures with serum from
the Komodo dragon results in 96.4% of the bacteria killed. However, mild heat treatment
(56oC, 30 min) of the serum resulted in only
4.0% antibacterial activity. Likewise, treatment
of the serum with 5 mM EDTA or 5 mM phosphate resulted in only 6.3% or 8.7% antibacterial activity, respectively.

Discussion

Eukaryotic organisms must continuously

defend themselves against infiltration and colonization by microorganisms. Host defense
occurs via complex mechanisms and can be
divided into two distinct, but interrelated,
types of responses: acquired immunity and
innate immunity. Acquired immunity requires
prior exposure to a specific antigen before a
full immunological assault can be established
by the host organism. In addition, the acquired
immune response is complex and can often
take several days to become fully activated. The
innate immune response comprises a significant portion of the immune system and acts as
an initial defense mechanism against microbial growth shortly after infection occurs.
These innate defense responses are activated
shortly after exposure and act to slow or stop
an infection in the initial stages so that the
acquired immune response can be initiated.
Also, the innate immune system functions in
the activation of the acquired immune system
by generating chemotactic factors and producing cytokines that initiate the development
and maturation of specific T-cell and B-cell
populations. Anecdotal evidence suggests that
Komodo dragons are resistant to microbial

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Two hundred L samples containing various

volumes (20-200 L) of Komodo dragon serum
in sterile microtiter plates were inoculated
with five L of bacterial culture from log phase
cultures and incubated for 24 h at 37oC. The
optical density of each sample was measured
at 0, 3, 6 12 and 24 h using a Biorad
Benchmark Plus microtiter plate reader at
430 nm.

The results in Figure 1 show that serum

from V. komodoensis inhibits the growth of
Gram negative bacteria. The serum inhibited
growth of E. coli in a volume-dependent manner (Figure 1A). At 24 hrs, 10% V. komodoensis
serum (v/v) produced a 44% decrease in E. coli
growth, while 25, 50, 75, and 100% serum
caused 58, 75, 93, and 98% decreases in
growth, respectively. By way of comparison,
incubation of 10% V. komodoensis serum with
Klebsiella oxytoca cultures resulted in 34%
growth inhibition at 24 hrs, while 25% serum
caused a 65% decrease in bacterial density
(Figure 1B). All serum volumes at, or above,
50% completely inhibited the growth of K. oxytoca cultures at 24 hrs. Serum from V. komodoensis (10% volume) inhibited Shigella
flexnerii growth by 95% at 24 hrs (Figure 1D),
and exhibited similar results for cultures of
Salmonella typhimurium (Figure 1E). Cultures
of Providencia stuarti growth were completely
obliterated (100%) inhibition by only 10%
serum (Figure 1C).
Figure 2 illustrates the effects of serum
from V. komodoensis on the growth of Gram
positive bacteria. Serum (10% volume) from V.
komodoensis inhibited the growth of
Staphylococcus aureus by 64% at 24 hrs, while
25, 50, 75, and 100% serum resulted in 68, 84,
94, and 93% reduction in growth, respectively
(Figure 2A). In contrast, 10% serum from V.
komodoensis inhibited the growth of
Streptococcus epidermitis cultures by 49%
(Figure 2B).
The bacterial-killing capacity of serum from
V. komodoensis for E. coli is displayed in Figure
3. A 20 L sample of positive control culture of
E. coli in log phase growth was found to have
16.40.4 million viable bacteria. In contrast,
the addition of 25% (v/v) of serum from V.
komodoensis to the bacterial growth medium
caused a significant decrease (P<0.05) to
5.22.1 million CFU. Further increases in
serum to 50% resulted in decreased bacterial
viability (1.30.2 million CFU). Because only
20 L of the bacterial culture were used for
colony determination, we calculated that one
L of serum from V. komodoensis has the
capacity to kill approximately 2.24 million E.
coli bacteria.
Figure 4 shows the results of a kinetic analysis of the ability of serum from V. komodoensis
to inhibit the growth of E. coli bacterial cultures. The antibacterial activity occurred
quickly, as 18% of the bacteria (2.95 million)
were killed within 5 min of exposure to serum
from V. komodoensis. Further incubation
resulted in 43% (7.05 million) of the bacteria
killed at 10 min of exposure, and the antibacterial action was maximal at 20 min as 68%
(11.2 million) of the E. coli were killed.

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Serum volume-dependent
antibacterial assays

Results

the attending veterinarian. Blood was collected

from three adults (20-81.5 kg) and five juveniles (1.5-6.2 kg), and the serum was pooled so
that average antibacterial values for this
species could be generated. The samples were
stored at -20oC until used for the antibacterial
assays. The collection of blood from these animals was conducted in accordance with the
animal care institutional policies of the
Houston and San Antonio Zoos.

[Microbiology Research 2013; 4:e4]

Table 1. Effects of heat treatment, protease

treatment, or metal chelators on the antibacterial activity of serum from V. komodoensis
against E. coli. The E. coli survival was determined by manual colony counts. The results
represent the means of four independent
determinations.
Treatment
None
Komodo dragon serum
Serum, 56C, 30 min
Serum + pronase
Serum + 5 mM EDTA
Serum + 5 mM phosphate

CFU Antibacterial
activity
12.7106
0.46106
12.2106
12.4106
11.9106
11.6106

96.4%
4.0%
2.4%
6.3%
8.7%

CFU, colony-forming units.

[page 17]

Article
to mice, particularly Pasteurella multocida,
supporting the idea that bite wounds inflicted
by Komodo dragons may induce systemic septicemia in their prey. However, it would be reasonable to expect that Komodo dragons would
have developed innate immune mechanisms
to combat possible infection by the bacteria in
their own saliva. In addition, antibodies
against P. multocida were found in the plasma

of the Komodo dragons,4 supporting the idea

that their immune systems are equipped to
deal with the heavy bacteria load present in
their saliva. Antibacterial activities of other
reptilian species have been assessed, particularly those of several crocodilian species.8,9
Merchant et al.8 showed that the bactericidal
capacity of serum from the American alligator
(Alligator mississippiensis) toward E. coli was

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infections. These animals often sustain serious injuries, including open wounds, due to
interspecies fighting in territorial disputes.7
Despite the propensity of dragons to resist
microbial infection, the mechanisms of immunity are not well-characterized.
Montgomery et al.4 isolated many bacterial
species from both captive and wild Komodo
dragons, some of which were extremely toxic

Figure 1. Effects of serum from V. komodoensis on growth of Gram negative bacteria. Culture tubes containing 2 mL of 0, 10, 25, 50,
75 or 100% serum from V. komodoensis. The cultures were incubated at 37C and their optical densities were measured (430 nm) at 0,
3, 6, 12, and 24 h post-inoculation. The results are expressed as the means S.D. for four independent determinations. A) Eschericia
coliform, B) Klebsiella oxytoca, C) Providencia stuartii, D) Shigella flexneri, E) Salmonella typhimurium.

[page 18]

[Microbiology Research 2013; 4:e4]

Article
described in this study could be a result of the
evolution of these ancient vertebrates under
the conditions of territorial aggression, feeding disputes, challenges for mating rights, etc.
Bull et al.11 reported that 70% of the feeding
activities of V. komodoensis on large prey items
occurred with multiple adult animals in close
proximity. The broad spectrum of oral bacteria
isolated from this species and the high bacterial load of their saliva, 4,11 would certainly be an
issue if bite injuries occurred during territorial battles. Therefore, it is not surprising that
these animals could have evolved strong
innate immunity defenses to deal with the
potential for serious infection due to their
aggressive lifestyles. It must be noted that the
animals used in this study were captive animals, and thus the immune system could

potentially be altered by chronic stress of captivity. However, chronic stress typically acts as
an immunosuppressive factor in vertebrates,12-17
and thus the antibacterial activity of the serum
from wild V. komodoensis could potentially be
underestimated in this study.
The antibacterial effects of Komodo dragon
serum is most likely due to an efficient serum
complement innate immune-defense. The fact
that mild heat treatment (30 min. at 56oC)
almost completely obliterated the observed
activities is an indication that serum complement activity is responsible for the antibacterial activities. It has been well-established that
serum complement proteins are extremely vulnerable to mild heat treatment, as some of the
key proteins components are thermally unstable.18 In addition, the antibacterial activity was
sensitive to pronase, indicating that at least
one of the components required for the
observed activity is proteinaceous in nature.
This idea is reinforced by the dependence of
the antibacterial activities on divalent metal
ions (Table 1). It has long been known that
divalent metals (magnesium and calcium) are
required for mammalian serum complement
activity.19-20 More recent studies have confirmed the requirement for calcium and magnesium for reptilian serum complement function.21 It is not surprising that V. komodoensis
would have serum complement proteins, as
most vertebrate, as well as many ancient invertebrates, exhibit complement activity.22
However, the strength with which these proteins kill bacteria is staggering. Before this
study, crocodilians were considered to have
among the most potent serum complement
defense systems.10 However, the results in this
study show that the potency of Komodo dragon
serum complement is much higher than that
of crocodilians.23 This study represents the
first report on innate immune activity of V.
komodoensis.

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54,000 bacteria/L. Other reptilians have been

shown to exhibit potent antibacterial activities, particularly crocodilian species. Merchant
et al.10 showed that serum from the American
alligator (Alligator missippiensis), when challenged with cultures of E. coli as low as 106/mL,
could not kill enough bacteria to make a statistical difference in the colony counts, relative to
untreated controls cultures. The capacity of
alligator serum to kill E. coli was approximately 54,000 bacteria/L. However, the results
shown in Figure 3 show that serum from V.
komodoensis has substantially higher activity
than that of Alligator mississippiensis. The
serum from V. komodoensis showed an approximate 40-fold increase in the capacity to kill E.
coli, relative to that of Alligator mississippiensis. The extreme antibacterial properties

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Figure 2. Effects of serum from V. komodoensis on growth of Gram positive bacteria.

Culture tubes containing 2 mL of 0, 10, 25, 50, 75 or 100% serum from Komodo dragons.
The cultures were incubated at 37C and their optical densities were measured (430 nm) at
0, 3, 6, 12, and 24 h post-inoculation. The results are expressed as the means S.D. for four
independent determinations. A) Staphylococcus aureus, B) Streptococcus epidermitis.

References

Figure 3. Antibacterial capacity of serum

from V. komodoensis against E. coli.
Different concentrations of Komodo dragon serum were incubated with E. coli cultures and plated on nutrient agar. CFUs
were determined after overnight incubation at 37C. The results are expressed as
the means S.D. for four independent
determinations.

Figure 4. Kinetics of antibacterial activity

of serum from V. komodoensis against E.
coli. Cultures of E. coli were incubated
with 25% (v/v) V. komodoensis. Aliquots
were removed at various time points and
plate on nutrient agar to determine the
bacterial survival. The results are expressed
as the means S.D. for four independent
determinations.

[Microbiology Research 2013; 4:e4]

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